Cellular senescence is a form of irreversible growth arrest, originally described for end-stage proliferative cells in culture, but known to be induced by several stimuli such as DNA damage, oxidative stress, chemotherapy and excess of mitotic stimuli such as oncogenic activation (Serrano et al 1997; Campisi 2001; Schmitt et al 2002). Cells enter in a stage of irreversible arrest, showing distinctive features, including enhanced beta-galactosidase activity and increased expression of key mediators including p53, promyelocitic leukemia protein (PML), p16INK4a and p19Arf (Serrano et al 1997; Narita et al 2003; Sharpless et al 2004). Although mostly studied in vitro, cellular senescence has been correlatively linked to the aging process at the level of the whole animal, thus implicating many of the factors that regulate senescence as contributing to organism aging (Sharpless and DePinho 2004, Campisi 2005).
Recent findings identify the p53-related protein p63, as a key molecule linking senescence and aging (Keyers et al 2005; Sommer et al 2006). Indeed, p63 heterozygous mice have a shortened life span and display features of accelerated aging. Both germline and somatic p63 deficient cells present enhanced expression of beta-galactosidase activity, PML and p16INK4a, demonstrating that p63 deficiency accelerated the aging phenotype (Keyers et al 2005). Beside the involvement in aging, the transcription factor p63 has an important role in development due to its ability to regulate epithelial proliferation, differentiation and cell fate. p63 KO mice exhibit several epithelial defects, including absence of skin, hair and ectodermal derivatives (Yang et al 1999; Mills et al 1999). Some reports indicate that p63 is essential for maintaining the stem cell pool of the epidermis (Seeno et al 2007). Recently, it has been reported that p63 is also regulated by microRNAs. In particular, it has been shown that miR-203, by targeting p63, controls the proliferative potential of basal layer keratinocytes (Lena et al 2008; Yi et al 2008), thus controlling the crucial transition among basal and spinous layer.
SirT-1 (or Sirt-1) is also a key molecule linking senescence and aging. It's a NAD+-dependent deacetylase that regulates gene expression by deacetylation of lysine residues on histones, transcription factor and co-factors. Cells with SirT-1 gain of function improves metabolic efficiency, promoting longevity and resistance to diseases (Haigis M C, Guarente L P. Mammalian sirtuins-emerging roles in physiology, aging, and calorie restriction. Genes Dev. 2006. 1; 20(21):2913-21). In addition it as been known that SirT-1 has a protective effects against endothelial dysfunction by preventing stress-induced senescence (Potente M, Ghaeni L, Baldessari D, Mostoslaysky R, Rossig L, Dequiedt F, Haendeler J, Mione M, Dejana E, Alt F W, Zeiher A M, Dimmeler S. SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev. 2007. 21(20):2644-58). Recently, it has been shown that SirT-1 is regulated by the microRNA miR-217 in endothelial cell senescent system (Menghini R, Casagrande V, Cardellini M, Martelli E, Terrinoni A, Amati F, Vasa-Nicotera M, Ippoliti A, Novelli G, Melino G, Lauro R, Federici M. MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation. 2009. 120(15):1524-32).
CDK6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb (retinoblastoma) kinase in the regulation of the G(1) phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation (Kohrt D M et al., Cycle. 2009 1; 8(17):2837-43; Grossel M J, et al., J Cell Biochem. 2006; 97(3):485-93). Cdk-6 has never been described before to be involved in senescence.
MicroRNAs (miRs) are single-stranded RNA molecules that regulate the expression of messenger mRNA. miRs are generated as pre-microRNA transcripts that are imported into the cytoplasm, where they are cleaved by Dicer, a ribonuclease III-like enzyme. In mammals Dicer plays important roles in cell differentiation and tissue morphogenesis and ablation of Dicer in mice induces embryonic lethality at developmental stage E6-E7 (Bernstein et al 2003). Ablation of Dicer in embryonic fibroblasts induces a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and adult skin (Mudhasani et al 2008). The mature miRs negatively regulate gene expression by targeting specific messenger mRNAs for cleavage or translation repression. Growing body of evidence suggests that they are involved in the control of a wide range of physiological pathways including cellular senescence.
It is thus desirable and important to provide products or active agents which prevent, reduce or even inhibit the cellular senescence, particularly the keratinocyte senescence.
The present invention thus provides a method for identifying such useful agents.